(recombine) DNA

ahemhootBiotechnology

Dec 5, 2012 (4 years and 8 months ago)

722 views

Recombinant DNA technology:

A series of procedures used to join together (recombine) DNA
segments. A recombinant DNA molecule is constructed (recombined) from segments from 2 or more
different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and
r
eplicate there, autonomously (on its own) or after it has become integrated into a chromosome.

Recombinant DNA

From Wikipedia, the free encyclopedia

Jump to:
navigation
,
search


Recombinant DNA

(rDNA) molecules are
DNA

sequences that result from the use of laboratory
methods (
molecular cloning
) to bring together genetic material from multiple sources, creating
sequences

that would not otherwise be found in biological organisms. Recombinant DNA is
possible because DNA molecules from all organisms share the same chemical structure; they
differ only in the sequence of nucleotides within that identical overall structure. Con
sequently,
when DNA from a foreign source is linked to host sequences that can drive
DNA replication

and
then introduced into a host organism, the foreign DNA is replicated a
long with the host DNA.

Recombinant DNA molecules are sometimes called
chimeric DNA
, because they are usually
made of material from two different species, like the mythological
chimera
.

The DNA sequences used in the construction of recombinant DNA molecules can originate from
any
species
. For example, plant DNA may be joined to bacterial DNA, or human DN
A may be
joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may
be created by the
chemical synthesis of DNA
, and inco
rporated into recombinant molecules.
Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be
created and introduced into any of a very wide range of living organisms.

Proteins that result from the expression of recombinant DNA

within living cells are termed
recombinant proteins
. When recombinant DNA encoding a protein is introduced into a host
organism, the recombinant protein will not necessarily be produced
[
citation needed
]
. Expression of
foreign proteins requires the use of specialized
expression vectors

and often necessitates
significant restructuring of the foreign coding sequence
[
citation needed
]
.

It is important to note that recombinant DNA differs fr
om
genetic recombination

in that the
former results from artificial methods in the test tube, while the latter is a normal biological
process that results in the
remixing of existing DNA sequences in essentially all organisms.

Creating recombinant DNA

Main article:
Molecular cloning



Construction of recombinant DNA, in which a
foreign DNA fragment is inserted into a plasmid vector. In
this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA
fragment.

Molecular cloning is the laboratory process used to create recombinant DNA.
[1]
[2]
[3]
[4]

It is one of
two widely
-
used methods (along with
polymerase chain reaction
, abbr. PCR) used to direct the
replication of any specific DNA sequence chosen by the experimentalist. The fundamental
difference between the two methods is that molecular cloning involves replication of t
he DNA
within a living cell, while PCR replicates DNA in the test tube, free of living cells.

Formation of recombinant DNA requires a
cloning vector
, a
DNA molecule that will replicate
within a living cell. Vectors are generally derived from
plasmids

or
viruses
, and represent
rel
atively small segments of DNA that contain necessary genetic signals for replication, as well
as additional elements for convenience in inserting foreign DNA, identifying cells that contain
recombinant DNA, and, where appropriate, expressing the foreign DN
A. The choice of vector
for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned,
and whether and how the foreign DNA is to be expressed.
[5]

In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps:
(1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation
of DNA to be cloned, (4) Creation of recombin
ant DNA, (5) Introduction of recombinant DNA
into the host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening
for clones with desired DNA inserts and biological properties.
[4]

These steps are described in
some detail in a related article (
molecular cloning
).

[
edit
]

Expression of recombinant DNA

Main article:
Gene expression

Following transplantation into the host organism, the foreign DNA contained within the
recombinant DNA construct may or may not be
expressed
. That is, the DNA may simply be
r
eplicated without expression, or it may be
transcribed

and
tra
nslated

so that a recombinant
protein is produced. Generally speaking, expression of a foreign gene requires restructuring the
gene to include sequences that are required for producing a
mRNA

mole
cule that can be used by
the host's
translational apparatus

(e.g.
promoter
,
translational initiation signal
, and
transcriptional
terminato
r
).
[6]

Specific changes to the host organism may be made to improve expression of the
ectopic gene. In addition, changes may be needed to the coding sequences as well
, to optimize
translation, make the protein soluble, direct the recombinant protein to the proper cellular or
extracellular location, and stabilize the protein from degradation.
[7]

[
edit
]

Properties of organisms containing recombinant DNA

In most cases
, organisms containing recombinant DNA have apparently normal
phenotypes
. That
is, their appearance, behavior and metabolism are usually unchanged, and the only way to
demonstrate the pr
esence of recombinant sequences is to examine the DNA itself, typically using
a polymerase chain reaction (PCR) test.
[8]

Significant exceptions exist, and are d
iscussed below.

If the rDNA sequences encode a gene that is expressed, then the presence of RNA and/or protein
products of the recombinant gene can be detected, typically using
RT
-
PCR

or
western
hybridization

methods.
[8]

Gross phenotypic changes are not the norm, unless the recombinant
gene has been chosen and modified so as to generate biological activity in the host organism.
[9
]

Additional phenotypes that are encountered include toxicity to the host organism induced by the
recombinant gene product, especially if it is
over
-
expressed

or expressed within inappropriate
cells or tissues.

In some cases, recombinant DNA can have deleterious effects even if it is not expressed. One
mechanism by which this happens is
insertional inactivation
, in which the rDNA becomes
inserted into a host cell’s gene. In some cases, researchers use this phenomenon to “
kn
ock out

genes in order to determine their biological function and importance.
[10]

Another mechanism by
which rDNA insertion into chromosomal DNA can affect gene expr
ession is by inappropriate
activation of previously unexpressed host cell genes. This can happen, for example, when a
recombinant DNA fragment containing an active promoter becomes located next to a previously
silent host cell gene, or when a host cell gen
e that functions to restrain gene expression
undergoes insertional inactivation by recombinant DNA.

[
edit
]

Applications of recombinant DNA technology

Recombinant DNA is widely used in
biotechnology
,
medicine

and
research
. Today, recombinant
proteins and other products that result from the use of rDNA technology are found in essentially
every western pharmacy, doctor's or veterinarian's office,

medical testing laboratory, and
biological research laboratory. In addition, organisms that have been manipulated using
recombinant DNA technology, and products derived from those organisms have found their way
into many farms,
supermarkets
,
home medicine cabinets

and even
pet shops
.

The most common application of recombinant DNA is in basic research, where it is important to
most current work in the biological and biomedical sciences.
[8]

Recombinant DNA is used to
identify, map and sequence genes, and to determine their function. rDNA probes are employed in
analyzing gene expression within individual cells, and throughout the tissues of whole
organisms. Recombinan
t proteins are widely used as reagents in laboratory experiments and to
generate antibody probes for examining protein synthesis within cells and organisms.
[2]

Many additional practical applications of recombinant DNA are found in human and veterinary
medicine, in agriculture, and in bioengineering.
[2]

Some specific ex
amples are identified below.



Recombinant human
insulin
. Recombinant insulin has almost completely replaced insulin obtained
from animal sources (e.g. pigs and cattle) for the treatment of insulin
-
dependent
diabetes
. A variety
of different rec
ombinant insulin preparations are in widespread use.
[11]

DrugBank entry
.



Recombinant human
growth hormone

(HGH, somatotropin). Growth hormone is administered to
patients whose pituitary glands generate insufficient quantities to support normal growth and
development. Before recombina
nt HGH became available, HGH for therapeutic use was obtained
from pituitary glands of cadavers. This unsafe practice led to some patients developing
Creu
tzfeldt
-
Jacob disease
. Recombinant HGH eliminated this problem, and is now used therapeutically.
[12]

It has
also been misused as a performance enhancing drug by
athletes and others.
[13]

DrugBank entry



Recombinant blood clotting
factor VIII
. Recombinant factor VIII is a blood
-
clotting protein that is
administered to patients with forms of the bleeding disorder hemophilia, who are unable to
produce factor VIII in quantities sufficient to support nor
mal blood coagulation.
[14]

Before the
development of recombinant factor VIII, the protein was obtained by processing large quantities of
human blood from multipl
e donors, which carried a very high risk of transmission of
blood borne
infectious diseases
, for example HIV and hepatitis B.
DrugBank entry



Recombinant
hepatitis B vaccine
. Prevention of
hepatitis B

infect
ion is controlled through the use
of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen
that is produced in yeast cells. The development of the recombinant subunit vaccine was an
important and necessary develo
pment because hepatitis B virus, unlike other common viruses such
as
polio virus
, cannot be grown
in vitro
.
Vaccine information from Hepatitis B Foundation



Diagnosis of infection with
HIV
. Each of the three widely
-
used methods for
diagnosing HIV infection

has been developed using recombinant DNA. The antibody test (
ELISA

or
western blot
) uses a
recombinant HIV protein to test for the presence of
antibodies

that the body has produced in
response to an HIV infection. The DNA

test looks for the presence of HIV genetic material using
reverse transcriptase polymerase chain reaction (
RT
-
PCR
). Development of the RT
-
PCR test was
made possible by the molecular cloning a
nd sequence analysis of HIV genomes.
HIV testing page
from US Centers for Disease Control (CDC)



Golden rice

is a recom
binant variety of rice that has been engineered to express the enzymes
responsible for
β
-
carotene

biosynthesis.
[9]

This variety of rice holds substantial promise for reducing
the incidence of
vitamin A deficiency

in the world's population.
[15]

Golden rice is not currently in use,
pending the resolution of intellectual property, environmental and nutritional issues.



Herbicide
-
resistant crops

Commercial varieties of important agricultural crops (including soy,
maize/corn, sorghum, canola, alfalfa and cotton) have been developed which incorporate a
recombinant gene that results in resistanc
e to the herbicide
glyphosate

(trade name
Roundup
), and
simplifies weed control by glyphosate application.
[16]

These crops are in common commercial use in
several countries.



Insect
-
resistant crops
. Bacillus thuringeiensis is a bacterium that naturally produces a protein (
Bt
toxin
) with insecticidal properties.
[15]

The bacterium has been applied to crops as an insect
-
control
strategy for many years, and this practice has been widely adopted in agriculture and gardening.
Recently, plants have been developed which expre
ss a recombinant form of the bacterial protein,
which may effectively control some insect predators. Environmental issues associated with the use
of these
transgenic

crops have not bee
n fully resolved.
[17]

The Basics of Recombinant DNA


So What Is rDNA?


That's a very good question! rDNA stands for recombinant DNA. Before

we get to the "r" pa
rt, we need to understand DNA. Those of you with

a background in biology probably know about DNA, but a lot of ChemE's haven't

seen DNA since high school biology. DNA is the keeper of the all the information

needed to recreate an organism. All DNA is ma
de up of a base consisting

of sugar, phosphate and one nitrogen base. There are four nitrogen bases,

adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen

bases are found in pairs, with A & T and G & C paired together. The sequence

of t
he nitrogen bases can be arranged in an infinite ways, and their structure is known as

the famous "double helix" which is shown in the image below. The sugar used in

DNA is deoxyribose. The four nitrogen bases are the same for all organisms. The

sequen
ce and number of bases is what creates diversity. DNA does not

actually make the organism, it only makes proteins. The DNA is transcribed

into mRNA and mRNA is translated into protein, and the protein then forms the

organism. By changing the DNA seq
uence, the way in which the protein is

formed changes. This leads to either a different protein, or an inactive protein.




Now that we know what DNA is, this is where the recombinant comes in.

Recombinant DNA is the general name for taking a piece of one DNA, and

and combining it with another strand of DNA. Thus, the name recombinant!

Recombinant DNA is also sometimes referred to as "chimera." By combining two or

more different strands of DNA
, scientists are able to create a new strand of DNA.

The most common recombinant process involves combining the DNA of two

different organisms.


How is Recombinant DNA made?


There are three different methods by which Recombinant DNA is made. They are

Transformation, Phage Introduction, and Non
-
Bacterial Transformation. Each

are described separately below.


Transformation

The first step in transformation is to select a piece of DNA to be inserted

into a vector. The second step is to cut that piece of

DNA with a restriction

enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a
selectable

marker which allows for identification of recombinant molecules. An antibiotic

marker is often used so a host cell without a
vector dies when exposed to a certain

antibiotic, and the host with the vector will live because it is resistant.


The vector is inserted into a host cell, in a process called transformation. One

example of a possible host cell is E. Coli. The host cell
s must be specially

prepared to take up the foreign DNA.


Selectable markers can be for antibiotic resistance, color changes, or any other

characteristic which can distinguish transformed hosts from untransformed hosts.

Different vectors have different

properties to make them suitable to different

applications. Some properties can include symmetrical cloning sites, size, and

high copy number.


Non
-
Bacterial Transformation

This is a process very similar to Transformation, which was described above. Th
e

only difference between the two is non
-
bacterial does not use bacteria such as E. Coli

for the host.


In microinjection, the DNA is injected directly into the nucleus of the cell being

transformed. In biolistics, the host cells are bombarded with hig
h velocity

microprojectiles, such as particles of gold or tungsten that have been coated

with DNA.


Phage Introduction

Phage introduction is the process of transfection, which is equivalent to transformation,

except a phage is used instead of bacteria.

In vitro packagings of a vector is used.

This uses lambda or MI3 phages to produce phage plaques which contain recombinants.

The recombinants that are created can be identified by differences in the

recombinants and non
-
recombinants using various selec
tion methods.



How does rDNA work?


Recombinant DNA works when the host cell expresses protein from the recombinant genes.

A significant amount of recombinant protein will not be produced by the host unless expression

factors are added. Protein expres
sion depends upon the gene being surrounded by

a collection of signals which provide instructions for the transcription and translation

of the gene by the cell. These signals include the promoter, the ribosome binding

site, and the terminator. Expressio
n vectors, in which the foreign DNA is inserted,

contain these signals. Signals are species specific. In the case of E. Coli, these

signals must be E. Coli signals as E. Coli is unlikely to understand the signals of

human promoters and terminators.


P
roblems are encountered if the gene contains introns or contains signals which act

as terminators to a bacterial host. This results in premature termination, and the recombinant

protein may not be processed correctly, be folded correctly, or may even be
degraded.


Production of recombinant proteins in eukaryotic systems generally takes place in

yeast and filamentous fungi. The use of animal cells is difficult due to the fact

that many need a solid support surface, unlike bacteria, and have complex grow
th

needs. However, some proteins are too complex to be produced in bacterium,

so eukaryotic cells must be used.



Why is rDNA important?


Recombinant DNA has been gaining in importance over the last few years, and

recombinant DNA will only become more important in the 21st century as genetic

diseases become more prevelant and agricultural area is reduced. Below are

some of the areas where Recombinant DNA will have an impact.



Better Crops (drought & heat resista
nce)



Recombinant Vaccines (ie. Hepatitis B)



Prevention and cure of sickle cell anemia



Prevention and cure of cystic fibrosis



Production of clotting factors



Production of insulin



Production of recombinant pharmaceuticals



Plants that produce their own

insecticides



Germ line and somatic gene therapy

Mutations: Types and Causes

The development and function of an organism is in large part controlled by genes. Mutations can lead to
changes in the structure of an encoded protein or to a decrease or comple
te loss in its expression.
Because a change in the DNA sequence affects all copies of the encoded protein, mutations can be
particularly damaging to a cell or organism. In contrast, any alterations in the sequences of RNA or
protein molecules that occur du
ring their synthesis are less serious because many copies of each RNA
and protein are synthesized.

Geneticists often distinguish between the
genotype

and
phenotype

of an organism. Strictly speaking, the
entire set of genes carried by an individual is its genotype, whereas the function and physical appearance
of an individual is referred to as its phenoty
pe
.

However, the two terms commonly are used in a more
restricted sense: genotype usually denotes whether an individual carries mutations in a single gene (or a
small number of genes), and phenotype denotes the physical and functional consequences of that
genotype.

The causes of mutations

Mutations happen for several reasons.

1. DNA fails to copy accurately

Most of the mutations that we think matter to evolution are "naturally
-
occurring." For example, when a cell divides, it
makes a copy of its DNA


and sometimes the copy is not quite perfect. That small difference from the original DNA
sequence is a mutatio
n.


2. External influences can create mutations

Mutations can also be caused by exposure to specific chemicals or radiation. These agents cause the DNA to break down.
This is not necessarily unnatural


eve渠n渠n桥 mos琠t獯la瑥d 慮d⁰物獴ine e湶i牯湭e湴s
Ⱐ,乁⁢re慫猠sow渮 乥ve牴heles猬
whe渠瑨e 捥ll⁲epai牳 瑨e D乁Ⱐ,琠tig桴 湯琠no 愠ae牦ec琠tob映t桥 牥pai爮r卯⁴ e 捥ll wo畬d e湤⁵ ⁷i瑨 D乁 slig桴ly
di晦eren琠瑨慮 瑨erigi湡l D乁 慮d⁨e湣eⰠ愠au瑡tio渮

Causes

Two classes of mutations are spontaneous
mutations (molecular decay) and induced mutations
caused by
mutagens
.

[
edit
]

Spontaneous mutation

Spontaneous mutations

on the molecular level can be caused by:
[23]



Tautom
erism



A base is changed by the repositioning of a hydrogen atom, altering the hydrogen
bonding pattern of that base resulting in incorrect base pairing during replication.



Depuri
nation



Loss of a purine base (A or G) to form an apurinic site (AP site).



Deamination



Hydrolysis changes a normal base to an atypical base containing a keto group in
place of the

original amine group. Examples include C → U and A → HX (hypoxanthine), which can
be corrected by DNA repair mechanisms; and 5MeC (5
-
methylcytosine) → T, which is less likely to
be detected as a mutation because thymine is a normal DNA base.



Slipped strand mispairing

-

Denaturation of the new strand from the template during replication,
followed by renaturation

in a different spot ("slipping"). This can lead to insertions or deletions.



A
covalent

adduct

between
benzo[
a
]pyrene
, the major
mutagen

in
tobacco smoke
, and DNA
[24]

[
edit
]

Induced mutation

I
nduced mutations

on the molecular level can be caused by:



Chemicals

o

Hydroxylamine

NH
2
OH

o

Base analogs

(e
.g.
BrdU
)

o

Alkylating agents (e.g.
N
-
ethyl
-
N
-
nitrosourea
) These

agents can mutate both replicating and
non
-
replicating DNA. In contrast, a base analog can only mutate the DNA when the analog is
incorporated in replicating the DNA. Each of these classes of chemical mutagens has certain
effects that then lead to transit
ions, transversions, or deletions.

o

Agents that form
DNA adducts

(e.g.
ochratoxin A

metabolites)
[25]

o

DNA
intercalating

agents (e.g.
ethidium bromide
)

o

DNA crosslinkers

o

Oxidative damage

o

Nitro
us acid converts amine groups on A and C to diazo groups, altering their hydrogen bonding
patterns which leads to incorrect base pairing during replication.



Radiation

o

Ultraviolet

ra
diation (nonionizing radiation). Two nucleotide bases in DNA


cytosine and
thymine


are most vulnerable to radiation that can change their properties. UV light can
induce adjacent
py
rimidine

bases in a DNA strand to become covalently joined as a
pyrimidine
dimer
. UV radiation, particularly longer
-
wave UVA, can also cause
oxidative damage to DNA
.
[26]

o

Ionizing radi
ation

o

Radioactive decay
, such as
14
C

in DNA



Viral

infections
[27]

DNA has so
-
called hotspots, where mutations occur up to 100 times more frequently than the
normal
mutation rate
. A hotspot can be at an unusual base, e.g.,
5
-
methylcytosine
.

Mutation rates

also vary across species. Evolutionary biologists
[
citation needed
]

have theorized

that
higher mutation rates are beneficial in some situations, because they allow organisms to evolve
and therefore adapt more quickly to their environments. For example, repeated exposure of
bacteria to antibiotics, and selection of resistant mutants, can

result in the selection of bacteria
that have a much higher mutation rate than the original population (
mutator strains
).

[
edit
]

Classification of mutation types



Illustrations of five types of chromosomal mutations.



Selection of dis
ease
-
causing mutations, in a standard table of the
genetic code

of
amino acids
.
[28]

[
edit
]

By effect on structure

The sequence of a gene can be altered in a
number of ways. Gene mutations have varying effects
on health depending on where they occur and whether they alter the function of essential
proteins. Mutations in the structure of genes can be classified as:



Small
-
scale mutations
, such as those affecting
a small gene in one or a few nucleotides, including:

o

Point mutations
, often caused by chemicals or malfunction of DNA replication, exchange a
single
nucleotide

for another.
[29]

These changes are classified as transitions or transversions.
[30]

Most common is the
transition

that exchanges a
purine

for a purine (A
↔ G) or a
pyrimidine

for
a pyrimidine, (C ↔ T). A transition can be caused by
nitrous acid
, base mis
-
pairing
, or
mutagenic base analogs such as
5
-
bromo
-
2
-
deoxyuridine (BrdU)
. Less common is a
transversion
, which exchanges a puri
ne for a pyrimidine or a pyrimidine for a purine (C/T ↔
A/G). An example of a transversion is
adenine

(A) being converted into a
cytosine

(C). A point
mutation can be reversed by another point mutation, in which the nucleotide is changed back
to its original state (true reversion) or by second
-
site reversion (a complementary mutation
elsewhere that results in regained gene fu
nctionality). Point mutations that occur within the
protein

coding region of a gene may be classified into three kinds, depending upon what the
erroneous
codon

codes for:



Silent mutations
: which code for the same
amino acid
.



Missense mutations
: which code for a different amino acid.



Nonsense mutations
: which code for a stop and can truncate the
protein
.

o

Insertions

add one or

more extra nucleotides into the DNA. They are usually caused by
transposable elements
, or errors during replication of repeating elements (e.g. AT repeats
[
citation
needed
]
). Insertions in the coding region of a gene may alter
s
plicing

of the
mRNA

(
splice site
mutation
), or cause a shift in the
reading frame

(
frameshift
), both of which can significantly
alter the gene product. Insertions can be reverted by excision o
f the
transposable element
.

o

Deletions

remove one or more nucleotides from the DNA. Like insertions, these mutations can
alter the
reading frame

of the gene. They are generally irreversible: though exactly the same
s
equence might theoretically be restored by an insertion, transposable elements able to revert
a very short deletion (say 1

2 bases) in
any

location are either highly unlikely to exist or do not
exist at all. Note that a deletion is not the exact opposite o
f an insertion: the former is quite
random while the latter consists of a specific sequence inserting at locations that are not
entirely random or even quite narrowly defined.



Large
-
scale mutations

in
chromosomal

structure, including:

o

Amplifications

(or
gene duplications
) leading to multiple copies of all chromosomal regions,
increasing the dosage of th
e genes located within them.

o

Deletions

of large chromosomal regions, leading to loss of the genes within those regions.

o

Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially bringing
together separate genes to form functionally distinct
fusion genes

(e.g.
bcr
-
abl
). These include:



Chromosomal translocations
: interchange of genetic parts from nonhomologous
chromosomes.



Interstitial deletions
: an intra
-
chromosomal deletion that removes a segment of DNA from
a single chromosome, thereby apposing previously distant genes. For example, cells
isolated from a human
astrocytoma
, a type of brain tumor, were found to have a
chromosomal deletion removing sequences between the "fused in glioblastoma" (fig) gene
and the receptor tyrosine kinase "ros", producing a fusion protein (FIG
-
ROS). The abnor
mal
FIG
-
ROS fusion protein has constitutively active kinase activity that causes oncogenic
transformation (a transformation from normal cells to cancer cells).



Ch
romosomal inversions
: reversing the orientation of a chromosomal segment.

o

Loss of heterozygosity
: loss of one
allele
, either by a deletion or
recombination

event, in an
organism that previously had two different alleles.

[
edit
]

By effect on function



Loss
-
of
-
function mutations

are the result of gene product having less or no function. When the
allele has a complete loss of function (
null allele
) it is often called an
amorphic

mutation
.
Phenotypes associated with such mutations are most often
recessive
. Exceptions are when the
organism is
haploid
, or when the reduced dosage of a normal gene product is not enough for a
normal phenotype (this is called
haploinsufficiency
).



Gain
-
of
-
function mutations

change the gene product such that it gains a new and abnormal
function. These mutations usually have
dominant

phenotypes.

Often called a
neomorphic

mutation.



Dominant negative mutations

(also called
antimorphi
c

mutations
) have an altered gene product
that acts antagonistically to the wild
-
type allele. These mutations usually result in an altered
molecular function (often inactive) and are characterised by a
dominant

or
semi
-
dominant

phenotype. In humans,
Marfan syn
drome

is an example of a dominant negative mutation occurring
in an
autosomal dominant

disease. In this condition, the defective glycoprotein product of the
fibrillin g
ene (FBN1) antagonizes the product of the normal allele.



Lethal mutations

are mutations that lead to the death of the organisms which carry the mutations.



A
back mutation

or
reversion

is a point mutation that restores the original sequence and hence the
or
iginal phenotype.
[31]

[
edit
]

By effect on fitness

In
applied genetics it is usual to speak of mutations as either harmful or beneficial.



A
harmful mutation

is a mutation that decreases the fitness of the organism.



A
beneficial mutation

is a mutation that increases fitness of the organism, or which promotes t
raits
that are desirable.

In theoretical population genetics, it is more usual to speak of such mutations as deleterious or
advantageous. In the
neutral theory of

molecular evolution
,
genetic drift

is the basis for most
variation at the molecular level.



A
neutral mutation

has no harmful or beneficial effect on the organism. Such mutations occur at a
steady rate, forming the basis for the molecular clock.



A
deleterious mutation

has a negative effect on the phenotype, and thus decreases the fitness of
the organism.



An
advant
ageous mutation

has a positive effect on the phenotype, and thus increases the fitness of
the organism.



A
nearly neutral mutation

is a mutation that may be slightly deleterious or advantageous, although
most nearly neutral mutations are slightly deleteriou
s.

In reality, viewing the fitness effects of mutations in these discrete categories is an
oversimplification. Attempts have been made to infer the distribution of fitness effects using
mutagenesis experiments or theoretical models applied to molecular seq
uence data. However, the
current distribution is still uncertain, and some aspects of the distribution likely vary between
species.
[22]

By inheritance



inheritable generic
in pro
-
generic tissue or cells on path to be changed to gametes.



non inheritable
somatic

(e.g., carcinogenic mutation)



non inheritable post mortem
aDNA

mutation in decaying remains.

By pattern of inheritance The human genome contains two copies of each gene


a paternal and a
maternal allele.



A
heterozygous mutation

is a mutation of only one allele.



A
homozygous mutation

is an identical mutation of both the paternal and maternal allel
es.



Compound heterozygous

mutations or a
genetic compound

comprises two different mutations in
the paternal and maternal alleles.
[32]



A
wildtype

or
homozygous non
-
mutated

organism is one in which neither allele is mutated. (Just
not a mutation)

[
edit
]

By impact on protein sequence



A
frameshift mutation

is a mutation caused by
insertion

or
deletion

of a number of nucleotides that
is not evenly divisible by three from a DNA sequence. Due to the triplet nature of gene expression
by
codons
, the insertion or deletion can disrupt the
reading frame
, or the grouping of the codons,
resulting in a completely different
translati
on

from the original.
[33]

The earlier in the sequence the
deletion or insertion occurs, the more altered the protein produced is.

In contrast, any insertion or deletion that is evenly divisible by three is termed an
in
-
frame
mutation



A
nonsense mutation

is a
point mutation

in a sequence of DNA that results in a premature
stop
codon
, or a
nonsense codon

in the transcribed mRNA, and possibly a truncated
, and often
nonfunctional protein product.



Missense mutations

or
nonsynonymous mutations

are types of
point mutations

where a single
nucleotide is changed to cause substitution of a different amino acid. This in turn can render the
resulting protein nonfunctional. Such mutations are responsible for diseases such as
Epidermolysis bullosa
,
sickle
-
cell disease
, and
SOD1

mediated
ALS

(
Boillé
e 2006
, p.

39).



A
neutral mutation

is a mutation that occurs in an amino acid codon

which results in the use of
a different, but chemically similar, amino acid. The similarity between the two is enough that
little or no change is often rendered in the protein. For example, a change from AAA to AGA will
encode
arginine
, a chemically similar molecule to the intended
lysine
. Neutral mutations occur
because of the
degenerate

nature of the genetic code.



Silent mutations

are mutations that do not result in a change to the amino acid sequence o
f a
protein. They may occur in a region that does not code for a protein, or they may occur within a
codon in a manner that does not alter the final amino acid sequence. The phrase
silent
mutation

is often used interchangeably with the phrase
synonymous mu
tation
; however,
synonymous mutations are a subcategory of the former, occurring only within exons. The name
silent could be a misnomer. For example, a silent mutation in the exon/intron border may lead
to
alternative splicing

by changing the splice site (
see
Splice site mutation
), thereby leading to a
changed protein.

[
edit
]

By inheritance ability



A mutation has caused this garden
moss rose

to produce flowers of different colors. This is a
somatic mutation that may also be passed on in the germ line.

In
multicellular organisms

wi
th dedicated
reproductive cells
, mutations can be subdivided
into
germ line mutati
ons
, which can be passed on to descendants through their reproductive
cells, and
somatic

mutations (also called acquired mutations),
[34]

which involve cells outside
the dedicated reproductive group and which are not usually transmitted to descendants. A
germline mutation gives rise to a
constitutional mutation

in the

offspring, that is, a mutation
that is present in every cell. A constitutional mutation can also occur very soon after
fertilisation
, or continue from a previous constitutional
mutation in a parent.
[35]

If the organism can reproduce
asexually

through mechanisms such as
cuttings

or
budding

the
distinction can become blurred. For example, plants can sometimes transmit somatic
mutations to their des
cendants asexually or sexually where flower buds develop in
somatically mutated parts of plants. A new mutation that was not inherited from either parent
is called a
de novo

mutation. The source of the mutation is unrelated to the
consequence
[
clarification needed
]
, although the consequences are related to which cells were
mutated.

[
edit
]

Special classes



Conditional mutation

is a mutation that has wild
-
type (or less severe) phenotype under certain
"permissive" environmental conditions and a mutant phenotype under certain "restrictive"
con
ditions. For example, a temperature
-
sensitive mutation can cause cell death at high
temperature (restrictive condition), but might have no deleterious consequences at a lower
temperature (permissive condition).

[
edit
]

Nomenclature

A committee of the Human Genome Variation Society (HGVS) has developed the standard
human sequence variant nomenclature,
[36]

which should be used by researchers and
DNA
diagnostic

centers to generate unambiguous mutation descriptions. In principle, this
nomenclature can also be used to describe mutations in other organisms. The nomenclature
specifies the type of mutation and base or amino acid changes.



Nucleotide substitution (e.g.

76A>T)
-

The number is the position of the nucleotide from the 5'
end, the first letter represents the wild type nucleotide, and the second letter represents the
nucleotide which replaced the wild type. In the given example, the adenine at the 76th positi
on
was replaced by a thymine.

o

If it becomes necessary to differentiate between mutations in genomic DNA, mitochondrial
DNA, and RNA, a simple convention is used. For example, if the 100th base of a nucleotide
sequence mutated from G to C, then it would be

written as g.100G>C if the mutation
occurred in genomic DNA, m.100G>C if the mutation occurred in mitochondrial DNA, or
r.100g>c if the mutation occurred in RNA. Note that for mutations in RNA, the nucleotide
code is written in lower case.



Amino acid subs
titution (e.g. D111E)


The first letter is the one letter code of the wild type
amino acid, the number is the position of the amino acid from the
N
-
terminus
, and the second
letter is
the one letter code of the amino acid present in the mutation. Nonsense mutations are
represented with an X for the second amino acid (e.g. D111X).



Amino acid deletion (e.g. ΔF508)


The Greek letter Δ (
delta
) indicates a deletion. The letter
refers to the amino acid present in the wild type and the number is the position from the N
terminus of the amino acid were it to be present as in the wild type.

[
edit
]

Harmful mutations

Changes in DNA caused by mutation can cause errors in
protein

seq
uence, creating partially
or completely non
-
functional proteins. To function correctly, each cell depends on thousands
of proteins to function in the right places at the right times. When a mutation alters a protein
that plays a critical role in the body,
a medical condition can result. A condition caused by
mutations in one or more genes is called a
genetic disorder
. Some mutations alter a gene's
DNA base sequence but do no
t change the function of the protein made by the gene. Studies
of the fly
Drosophila melanogaster

suggest that if a mutation does change a protein, this will
probably be harmful, with about 70 percent of these mutations having damaging effects, and
the remainder being either neutral or weakly beneficial.
[37]

However, studies in
yeast

have
shown that only 7% of mutations that are not in genes are harmful.
[38]

If a mutation is present in a
germ cell
, it can give rise to offspring that carries the mutation in
all of its cells. This is the case in
hereditary d
iseases
. On the other hand, a mutation may
occur in a
somatic cell

of an organism. Such mutations will be present in all descendants of
this cell within the same organism, and cert
ain mutations can cause the cell to become
malignant, and thus cause
cancer
.
[39]

Often, gene mutations that could cause a genetic disorder are repaired by the
DNA repair

system of the cell. Each cell has a number of pathways through which enzymes recognize
and repa
ir mistakes in DNA. Because DNA can be damaged or mutated in many ways, the
process of DNA repair is an important way in which the body protects itself from disease.

[
edit
]

Beneficial mutations

Although mutations that change protein sequences are predominantly harmful to an
organism; on occasion, the effect can be neutral or positive in a given environment. In this
case, the muta
tion may enable the mutant organism to withstand particular environmental
stresses better than wild
-
type organisms, or reproduce more quickly. In these cases a
mutation will tend to become more common in a population through
natural selection
.

For example, a specific 32
base pair

deletion in human
CCR5

(
CCR5
-
Δ32
) confers
HIV

resistance to
homozy
gotes

and delays
AIDS

onset in
heterozygotes
.
[40]

The CC
R5 mutation
is more common in those of European descent. One possible explanation of the
etiology

of
the relatively high frequency of CCR5
-
Δ32 in the European population is that it conferr
ed
resistance to the
bubonic plague

in mid
-
14th century Europe. People with this mutation were
more likely to survive infection; thus its frequency in the population increased.
[41]

This
theory could explain why this mutation is not found in southern Africa, where the bubonic
plague never reached. A newer theory suggests that the
selective pressure

on the CCR5 Delta
32 mutation was caused by
smallpox

instead of the bubonic plague.
[42]

Another example is
Sickle cell disease
, a blood disorder in which the body produces an
abno
rmal type of the oxygen
-
carrying substance
hemoglobin

in the
red blood cells
. One
-
third
of all
indigenous

inhabitants of
Sub
-
Saharan Africa

carry the gene,
[43]

because in areas where
malaria is common, there is a
survival value

in carrying only a single sickle
-
cell gene (
sickle
cell trait
).
[44]

Those with only one of the two
a
lleles

of the sickle
-
cell disease are more
resistant to malaria, since the infestation of the malaria plasmodium is halted by the sickling
of the cells which it infests.

[
edit
]

Prion mutation

Prions

are proteins and do not contain genetic material. However, prion replication has been
shown to be subject to mutation and
natural selection

just like other forms of replication.
[45]